Notch signaling is an important form of inter-cellular communication with a key role in cell-fate determination and differentiation (Artavanis-Tsakonas et al., 1999). The biological outcome of activation or suppression of this pathway is highly context-dependent. In many mammalian systems, Notch signaling enhances stem cell potential and suppresses differentiation, while in others, notably keratinocytes, it exerts an opposite role. Our main working hypothesis is that this pro-differentiation function of Notch in keratinocytes depends on a cell-type specific integration with other key regulatory pathways. Signaling mechanisms involved in growth/differentiation control of human and mouse keratinocytes, including their Notch response, are only partially overlapping. Therefore, in our future work, we will focus preferentially on human cells, utilizing the mouse system whenever necessary and as a point of reference. Notch signaling proceeds through a "canonical" pathway involving transcriptional activation of the CBF-1/Maml_1 complex and a less defined "non-canonical" pathway independent of CBF-1/Maml_1 function. We will address the following aims : 1) We will test the hypothesis that the "canonical" Notch pathway promotes keratinocyte commitment to differentiation through a cross-talk with signaling by small Rho GTPases. In particular, we have found that the Notch/CBF1 pathway is involved in cell type specific negative regulation of three key effectors of small Rho GTPases : the ROCK1, 2 and MRCKa kinases. Accordingly, we will assess whether down-modulation of these molecules is a key event in the Notch response of keratinocytes, and whether these kinases in turn control Notch signaling in these cells 2) We will test the hypothesis that the "non-canonical" Notch pathway plays a parallel important function in keratinocyte growth/differentiation control through an interplay with Interferon Response Factors and in particular IRF6, a transcription factor with an essential specific role in keratinocytes. We will evaluate the involvement of Notch signaling, in connection with IKKa, another selective regulator of keratinocyte differentiation, in control of IRF6 expression. Concomitantly, we will assess the impact of IRF6 and other IRFs on the Notch-response of keratinocytes, with p63, a key transcription factor for these cells, as an important endpoint. 3) We will test the hypothesis that integration of the above mechanisms plays a key role in long term control of self-renewing stem cell populations, and tumorigenesis. By both in vitro and in vivo assays, we will assess the balance between self-renewing and reversibly versus irreversibly committed populations. Concomitantly, we will determine the role of the above pathways in control of keratinocyte tumor development, by skin reconstitution / grafting experiments with genetically modified keratinocytes.